Clinical and histopathological study of renal biopsy in Nepalese children: A single center experience

Background Glomerular diseases are important causes of morbidity and mortality among children in developing countries. Renal biopsy is the gold standard for determining histological diagnosis, prognosis, and therapy options. This study aimed to investigate the clinical, histopathological, and direct immunofluorescence findings of renal biopsy results in Nepalese children under 18 years old. Methods In this retrospective cross-sectional study, the available data from children who had undergone kidney biopsy between 2016 and the end of 2020 were evaluated. Demographic data, indications of biopsy, histopathologic findings, and direct immunofluorescence findings were collected and presented. Results The mean age of the patients was 12.14 ± 4.07 years. Male/female ratio was 1:1. The majority of biopsy cases were aged between 11–15 years of age. The most common indication for biopsy in children was nephrotic syndrome (68.25%). Lupus nephritis with 28 cases (22.22%) had the highest frequency in overall renal biopsies. Minimal change disease (MCD) with 22 cases (17.46%) followed by Ig A nephropathy with 16 cases (12.69%) were the most frequent primary glomerulonephritis. Lupus nephritis showed full house positivity, and MCD showed full house negativity in all Direct immunofluorescence (DIF) parameters, whereas immunoglobulin A nephropathy showed 100% positivity in Ig A in DIF. Conclusions Nephrotic syndrome was the most common indication for renal biopsy. The most common primary glomerulonephritis was MCD, while secondary glomerulonephritis was lupus nephritis. Clinical data, light microscopy, and direct DIF played an integral role in the overall final diagnosis.


Introduction
Glomerular diseases are significant causes of morbidity and mortality and a considerable burden on health services in developing countries. Diagnosis and classification of glomerular disease pose a significant challenge to the pathologist due to the complexity and variety of these lesions [1].
Many children with kidney disease do not need a kidney biopsy for diagnosis. However, renal biopsy remains an essential tool for establishing a histological diagnosis, determining disease severity and prognosis, planning, and monitoring treatment modalities in some patients. Nephrotic syndrome with steroid resistance, steroid dependence or atypical features, recurrent hematuria, systemic lupus erythematous nephritis (SLE), Henoch Schonlein purpura nephritis (HSP), and unexplained acute kidney injury (AKI) are few indications of renal biopsy in children [2].
The knowledge of epidemiology, a pattern of renal disease, and histopathological association with clinical features in particular regions are different worldwide due to differences in race, socio-economical gaps, and guidelines for performing kidney biopsies in children [3].
There is a scarcity of data regarding the prevalence of glomerular diseases in children and their association with histopathological findings in Nepal. Therefore, this study was conducted to describe the spectrum of glomerular diseases in pediatric age groups, indications for renal biopsy, their histopathological appearance, and their clinical correlation based on light microscopic and immunofluorescence findings.

Patients
This was a retrospective study of renal biopsy samples received in the Pathology Department of Grande International Hospital of children aged less than 18 years who underwent percutaneous renal biopsies between January 2016 and December 2020. All biopsies were performed by nephrologists under ultrasound guidance. Two tissue cores were collected, one for light microscopy, which was kept in 10% neutral buffered formalin, and another core for immunofluorescence, which was held in normal saline and then transported to our laboratory. Renal biopsies were examined using light microscopy and DIF.

Ethical consideration
The study was approved by the Institutional Review Committee of Grande International Hospital and carried out in accordance with the Declaration of Helsinki. The need for informed consent was waived by "The Institutional Review Committee of Grande International Hospital (IRC-GIH)" IRB due to the retrospective nature of the study.

Data collection
Medical records of all children who underwent renal biopsy were retrieved from the medical record database and analyzed. The following data for each patient: name, age, sex, indication of kidney biopsy, histopathological findings, histopathological diagnosis, and laboratory investigations such as serum creatinine, 24-hour urinary protein, urine microscopy, anti-double- stranded DNA antibody, antinuclear antibody (ANA), complements levels 3 and 4 (C3, C4) were noted.

Histopathological analysis
For the light microscopic study, tissue processing was performed from formalin-fixed and paraffin-embedded (FFPE) tissues. Tissue sections were cut at 4μm thickness and staining was done using hematoxylin and eosin (HE), periodic acid-Schiff (PAS), Congo red stain, and Masson's Trichrome stain. For DIF, Dako reagent with appropriate dilution was prepared and a fresh frozen tissue specimen was cut in a cryostat. Air dry slides were stained for 20 minutes. It was prewashed in Phosphate buffered saline (PBS) for 5-10 minutes. 50-100 μl of working dilution of the Fluorescein isothiocyanate (FITC) conjugated antibody was applied. The slide was incubated at room temperature for 30 minutes in a moist chamber and was washed in Phosphate buffered saline (PBS) for 3 changes, 5 minutes each. Cover slip was used with glycerol-based mounting media. Immunoglobulins and complement analyses were done using an immunohistochemical antibody panel to immunoglobulins G, M, A (IgG, IgM, IgA), C3, complement 1 q (C1q), and also to kappa and lambda light chains. All kidney biopsy specimens obtained were prepared as per the standard protocol and examined by renal pathologists using Leica DM 1000 LED microscope. Analysis included light microscopy (LM) and immunofluorescence (IF). However, electron microscopy (EM) was not systematically performed, as this facility was not available in our institution.

Statistical analysis
Data analysis was done with SPSS 17.0 and percentages and proportions of indications, demographic parameters, histopathological findings, histopathological diagnosis, and their clinical correlation were calculated.

Demographic data and clinical presentation
A total of 126 patients were biopsied during the study period of five years. Of the 126 cases, 63 (50%) were boys and 63 (50%) were girls. The mean age was 12.14 ± 4.07 years and the age ranged from 1 year to 18 years as shown in Fig 1. Females had a mean age of 12.90 ± 3.81 years and males 11.38 ± 4.21years. The most common clinical indications for renal biopsy were nephrotic syndrome 36 (28.57%), followed by nephritic syndrome 25 (19.8%) and Systemic Lupus Erythematosus (SLE) 23 (18.25%), respectively. All these cases of SLE were positive for ANA and dsDNA. The frequency of causes of renal biopsies in the studied children is illustrated in Fig 2. Of 36 patients with nephrotic syndrome, 24 (68.25%) were steroid-resistant, 11 (30.55%) were steroid-sensitive, and 1 (2.78%) was frequently relapsing.
ANA/ds-DNA was done in 54 cases, of which 23 (42.59%) cases were positive.

Histopathological diagnoses
The results showed that primary glomerular disease was seen in 86 cases (68.25%) of all 126 biopsied patients. It was followed by secondary glomerular disease and hereditary glomerular disease which were seen in 37 cases (29.36%) and three cases (2.39%), respectively. The histopathological findings in kidney biopsied patients are shown in Table 1.
Most of the patients 73(57.93%) were in the age group 11 to 15 years. Age-wise breakdown of histopathological diagnoses are shown in Table 2.
Tubules. There was acute mild injury in renal tubules in 89 (70.63%) cases, acute moderate injury in 29 (23.01%) cases, acute severe injury in 3 (2.38%) cases, and 5 (3.96%) cases were unremarkable. According to the modified NIH activity chronicity index in lupus nephritis, Activity index <12 was in 22 (78.6%) cases and activity index >12 was seen in 6 (21.4%) cases. Similarly, Chronicity Index <4 was seen in 26 (92.8%) cases, and the chronicity Index >4 was seen in 2 (7.2%) cases. These findings along with the activity chronicity index of lupus nephritis are illustrated in Fig 4A and 4B.
The pattern of Glomerular disease according to clinical indications is shown in Table 3 On Direct Immunofluorescence (DIF), Lupus nephritis showed full house positivity in almost all cases with maximum positivity in Ig G (82.14%) followed by C3 (78.57%) and C1q (75%) respectively. (Fig 8) Ig A nephropathy cases showed strong positivity in Ig A (100%) and C3 (37.5%) (Fig 9). In contrast, MCD cases showed negativity in all DIF parameters.
The pattern of Glomerular disease according to direct immunofluorescence microscopy is shown in Table 4.

Demographic data and clinical presentation
Renal biopsy is a safe procedure in children and occupies a fundamental position in the management of kidney diseases [3]. In our study, there was an equal predominance of males and females. However, in other studies done by various authors, there was a slight male predominance [5,[7][8][9][10][11] except in a study done by Momtaz HE et al in which there was female predominance [4]. The mean age at biopsy was 12.14 ± 4.07 years, and age ranged from 1 to 18 years, which was similar to studies done in other countries [4][5][6][7][8][9][10][11]. In our study, most patients were in the age group of 11-15 years which was similar to the study done by Shrestha et al. [2].

Clinical indications
In our study, the nephrotic syndrome was the most common indication for biopsy in children, which was similar to other research [4][5][6][7][8][9][10][11]. Similarly, in a study done by Wannous H et al, the steroid-resistant nephrotic syndrome was the most common cause of renal biopsy after treatment failure [3]. However, in other studies, steroid-sensitive cases were most commonly biopsied. [4,7,11]. Glomerular disease according to clinical indications showed that Minimal change disease (52.7%) and FSGS (22.22%) were the two most common diseases presenting with nephrotic syndrome whereas Lupus Nephritis (95.65%) was the predominant disease in suspected SLE. Post-infectious GN (32%) and MPGN (16%) were the two important diseases presenting with nephritic syndrome. These findings were similar to the study done by Alyousef A et al [12].
In our study, ANA/ds-DNA was done in 54 cases of which 23 (42.59%) cases were positive and all were cases of Lupus nephritis. In a similar study done by Karki et al. on 38 patients with SLE, Antinuclear antibody (ANA) was positive in all 38 (100%), Anti-dsDNA seen in 18 (47.4%) cases [13]. Pediatric renal biopsy studies showing demographic details, indications, and histopathological spectrum are illustrated in Table 5.
SLE was the most commonly reported pathologic diagnosis in secondary glomerulonephritis with a frequency of 22.22%. A higher frequency of childhood-onset SLE has been reported in Asians in various studies [15,16]. In our study, it was more common in girls in an age group of 11-15 years. All of them were dsDNA/ANA positive.
MCD was the most common histopathological diagnosis in primary glomerulonephritis with a prevalence of 17.46%. It was more common among boys. They were all prevalent in the age group 11-15 years of age group. Most of them presented with nephrotic syndrome. MCD was one of the major causes of primary glomerular disease in children in Asian populations [17]. Similarly, a study was done by Priyadarshini et al [7] and Alyousef et al. [12] showed MCD as the commonest pathological diagnosis among children accounting for 47.63% and 35.5% respectively. The incidence of MCD cases in our study may have been underestimated as many patients responded to steroid treatment and were not biopsied. Most of our cases were steroid-resistant during the time of biopsy.
IgA nephropathy was the second most common diagnosis in primary glomerulonephritis with a prevalence of 12.69%. It was more common among boys in an age group of 11 to 15 years. Clinically, patients presented with nephrotic as well as nephritic syndrome, non-nephrotic proteinuria, or isolated hematuria. Similarly, other studies showed that IgA nephropathy was more prevalent among children in Asian populations [18][19][20]. Studies done by Arapovic et al [5] in Croatia and Lee et al. [10] in South Korea also showed IgA nephropathy as the most common diagnosis in histopathology in pediatric renal biopsies with 24.1% and 27.9% respectively.
FSGS was the third most common histopathological diagnosis in our study with a prevalence of 10.31% more commonly seen in males and age group of 11-15 years. Clinically, they presented more with features of nephrotic syndrome. Similarly, in studies done by Momtaz et al [4] and Moorani et al. [9], FSGS was the most common diagnosis with 32.7% and 29.66% respectively.

Histopathological lesions in lupus nephritis
The role of the International Society of Nephrology and the Renal Pathology Society (ISN/ RPS) 2016 classification of Lupus nephritis is to bring standardization and uniformity in the reporting of all renal pathology reports of Lupus nephritis, better clinical-pathological correlation, and its therapeutic and prognostic implications [21,22].
According to the International Society of Nephrology and the Renal Pathology Society (ISN/RPS) 2016 classification of Lupus nephritis in our study, Class IV (57.1%), followed by Class III (35.7%) were most common. Activity index <12 in 78.6% and chronicity index <4 in 92.8% were primarily observed in our study. In various studies, the most common histology of lupus nephritis in Asian children was Class IV lupus nephritis (39.4-54%), which is also true in other Western countries such as the United States (38%) and Canada (46%) [23]. Class IV Lupus nephritis is the most common Lupus nephritis in South East Asian countries as shown in Table 6 [2,9,12,[23][24][25][26][27][28][29].

Histopathological findings in IgA nephropathy
The updated Oxford Classification (MEST-C) 2016 has therapeutic and prognostic implications [34,35]. According to the updated Oxford Classification (MEST-C) 2016, in our study, the most common lesions were M1, E, S, T, and C. However, in a study done by Moriyama [34] et al., M, and S1 were more common than M1 and S, but E, T, and C were more in their study, which was similar to our research.

Direct immunofluorescence findings
Light microscopy gives the morphological pattern only, so to be more precise, we need to correlate light microscopy findings with clinical features; immunofluorescence and electron microscopy [36]. Direct immunofluorescence microscopy was done in all renal biopsies. The  Table 7 [4,37]. On DIF, Lupus nephritis showed full house positive in almost all cases with maximum positivity in Ig G (82.14%) followed by C3 (78.57%) and C1q (75%) respectively. Ig A nephropathy showed strong positivity to Ig A (100%) and C3 (37.5%). In contrast, MCD showed negativity in all DIF parameters. Moreover, a study done by Buch CA et al. also showed full house

Conclusion
• The most common indication of pediatric renal biopsy was nephrotic syndrome.
• MCD was the most frequent diagnosis among Primary GN.
• Lupus nephritis was the most common overall diagnosis and Secondary GN.
• Ig A nephropathy was the second most common diagnosis in Primary GN.
• Lupus nephritis showed full house positivity, minimal change disease showed full house negativity in all DIF parameters, whereas immunoglobulin A nephropathy showed 100% positivity in Ig A in DIF.
• Clinical data, light microscopy, and DIF play an integral role in the overall final diagnosis.
• The data of this study is based on a single center experience and do not represent prevalence of kidney disease in children in our country.

Limitations of the study
• The unavailability of electron microscopy for the examination of equivocal cases is a challenge to a developing country like Nepal.
• Most of the patients in our case were from rural areas and presented to the hospital at the late presentation of the disease in severe form with full-blown features.
• Minimal change disease cases were already given steroid treatment before biopsy. Many of them were steroid-resistant during the time of biopsy. Therefore the incidence of MCD may be underestimated.